**6. Chlorophyllin**

Chlorophyll was detected as an antimutagenic agent since the early 80s. However, very soon afterwards most research on the matter was focused on the effect of chlorophyllin (CHL) (Figure 4), mostly because this chemical is a water-soluble, sodium and copper salt, chlorophyll derivative. In regard to its protective effect on AFB1 damage, one of the first studies with CHL was made by quantifying the number of revertants of specific base-pair mutants in *Neurospora crassa* (Whong et al., 1988). The study showed strong inhibition of the antimutagen on the AFB1-mutagenicity determined in growing cultures of the mold, a result which prompted the group to continue their studies but now in *Salmonella typhimuriun*, strain TA98, as a model (Whong et al., 1988). The authors used the plateincorporation test in this bacteria and found a concentration-dependent inhibition of AFB1 mutagenicity when the cells were treated with the tested substances, concurrently; in their assay they observed that 860 nmol/plate of CHL completely abolished the mutagenicity of the toxin. However, when other approaches were tested, negative effects were detected; therefore, the authors suggested that CHL acted before the mutagen entered the bacterial cell by suppressing metabolic activation or by scavenging the mutagen.

The preventive capacity of CHL on the AFB1-DNA damage was confirmed by means of the arabinose-resistant *Salmonella* forward mutation assay (Warner et al., 1991); the results of the study were obtained in a preincubation test, and showed an inhibitory effect of CHL with and without the addition of S9 mix. With 2.5 mg/plate or less, the authors reported an almost complete inhibition of the aflatoxin mutagenicity.

In the following years, a number of studies were made, particularly in rainbow trout but also in microorganisms. Their purpose was to confirm the CHL antimutagenic potential by means of various models, as well as to investigate the involved mechanism(s) of action and to evaluate its cancer chemopreventive capacity (Breinholt et al., 1995a, 1995b; Dashwood et al., 1991). Results of these studies confirmed the in vitro efficiency of CHL when liver microsomes were added to the system, not only against the damage induced by AFB1, but also on the mutagenic effect induced by its precarcinogenic metabolite, 8,9 epoxide. In rainbow trout, the authors found an inhibitory effect of CHL on the precarcinogenic hepatic DNA adduction induced by AFB1, as well as a significant lowering in the number of liver tumors. In agreement with this finding, a study made in rat concluded that a concurrent administration of both compounds engendered an important reduction in the level of liver AFB1-DNA adducts (Kensler et al., 1998a), and another study showed a significant inhibitory effect of CHL (60%) on the morphological transformation of BALB/3T3 cells (Wu et al., 1994). In regard to the mechanism of action, a strong complex formation between mutagen and antimutagen was proposed.

Fig. 4. Chemical structure of chlorophyllin

262 Aflatoxins – Detection, Measurement And Control

ionization/mass spectrometry (Sharma & Farmer, 2004; Wang et al., 2008).

Chlorophyll was detected as an antimutagenic agent since the early 80s. However, very soon afterwards most research on the matter was focused on the effect of chlorophyllin (CHL) (Figure 4), mostly because this chemical is a water-soluble, sodium and copper salt, chlorophyll derivative. In regard to its protective effect on AFB1 damage, one of the first studies with CHL was made by quantifying the number of revertants of specific base-pair mutants in *Neurospora crassa* (Whong et al., 1988). The study showed strong inhibition of the antimutagen on the AFB1-mutagenicity determined in growing cultures of the mold, a result which prompted the group to continue their studies but now in *Salmonella typhimuriun*, strain TA98, as a model (Whong et al., 1988). The authors used the plateincorporation test in this bacteria and found a concentration-dependent inhibition of AFB1 mutagenicity when the cells were treated with the tested substances, concurrently; in their assay they observed that 860 nmol/plate of CHL completely abolished the mutagenicity of the toxin. However, when other approaches were tested, negative effects were detected; therefore, the authors suggested that CHL acted before the mutagen entered the bacterial cell by suppressing metabolic activation or by scavenging the

The preventive capacity of CHL on the AFB1-DNA damage was confirmed by means of the arabinose-resistant *Salmonella* forward mutation assay (Warner et al., 1991); the results of the study were obtained in a preincubation test, and showed an inhibitory effect of CHL with and without the addition of S9 mix. With 2.5 mg/plate or less, the authors reported an

In the following years, a number of studies were made, particularly in rainbow trout but also in microorganisms. Their purpose was to confirm the CHL antimutagenic potential by means of various models, as well as to investigate the involved mechanism(s) of action and to evaluate its cancer chemopreventive capacity (Breinholt et al., 1995a, 1995b; Dashwood et al., 1991). Results of these studies confirmed the in vitro efficiency of CHL when liver microsomes were added to the system, not only against the damage induced by AFB1, but also on the mutagenic effect induced by its precarcinogenic metabolite, 8,9 epoxide. In rainbow trout, the authors found an inhibitory effect of CHL on the precarcinogenic hepatic DNA adduction induced by AFB1, as well as a significant lowering in the number of liver tumors. In agreement with this finding, a study made in rat concluded that a concurrent administration of both compounds engendered an important reduction in the level of liver AFB1-DNA adducts (Kensler et al., 1998a), and another study showed a significant inhibitory effect of CHL (60%) on the morphological transformation of BALB/3T3 cells (Wu et al., 1994). In regard to the

almost complete inhibition of the aflatoxin mutagenicity.

**6. Chlorophyllin** 

mutagen.

body fluids is highly sensitive and specific to determine the effect of the studied xenobiotic. DNA adducts have been clearly shown to be relevant to the disease process in prospective studies (Bonassi & Au, 2002). In relation to our present review, the adducts 8,9-dihydro-8-(N(7)-guanyl-)-9-hydroxyaflatoxin, as well as the AFB1 formamidopyrimidine compound, among others, are thought to be involved in the mutations caused by AFB1. The detection of these compounds can be made in various organs, as well as in serum and urine, by means of a variety of methods that include HPLC, ELISA, accelerator mass spectrometry, and liquid chromatography/electrospray

> Based on the described positive experimental studies, efforts were initiated to determine the CHL chemopreventive capacity in humans. Qidong, People's Republic of China is a high risk region for hepatocellular carcinoma probably related with the consumption of AFB1 contaminated food; here, a randomized, double-blind, placebo-controlled trial was made to determine whether CHL administration altered the disposition of aflatoxin. CHL consumption at each meal led to an overall 55 % reduction in a median urinary level of the biomarker, aflatoxin-N7-guanine, compared with individuals taking placebo (Egner et al., 2001). The determined adduct biomarker derives from the carcinogenic metabolite, aflatoxin 8,9-epoxide; thus, the authors suggested that prophylactic interventions with CHL or supplementation of diets with chlorophyll rich foods may be useful to prevent the development of hepatocellular carcinoma or other environmentally induced cancers. This type of studies was supported by reports on the experimental effect of CHL in rats (Simonovich et al., 2007). The authors observed the inhibition of AFB1-albumin adducts and of AFB1-N7-guanine adducts, as well as the inhibition of AFB1 uptake when quantified in feces, besides a decrease in the number of colonic aberrant crypt foci induced by the aflatoxin. However, no modification in the activity of phase II enzymes was found.

> In summary, a number of in vitro and in vivo studies have supported the antigenotoxic and chemopreventive capacity of CHL against the damage induced by AFB1, activities which can be related with the formation of a strong non-covalent complex, although additional mechanisms, such as its antioxidant potential, cannot be discarded. However, a word of caution about safety in using CHL is pertinent in light of the negative or controversial results that have been published in regard to the compound: for example, its effect as both inhibitor or promoter of genetic damage depending on the tested approach (Cruces et al.,

Aflatoxin B1 - Prevention of Its Genetic Damage by Means of Chemical Agents 265

controlled, double-blind studies of people who had ingested AFB1 via their usual diet. For the rationale of the study the authors considered that the AFB1 epoxides are substrates for glutation S transferase (GST), an enzyme which catalyzes the conjugation of the epoxide with reduced glutation, thus mitigating the formation of DNA adducts. In fact, the results showed an increase in the activity of GST related with a sustained low dose of OL (125 mg/day), also yielding a high level of the AFB1 urinary metabolite, mercapturic acid; however, an intermittent, high dose of OL inhibited the activation of phase I enzymes, as reflected by a lowering in the excretion of the metabolite AFBm1. Nevertheless, the results of another study made in the same population suggested that prevention of oxidative DNA damage by OL was not a relevant mechanism to explain its effect against AFB1 (Glintborg et

The above-mentioned reports, as well as others, have suggested that the major preventive action of OL is through the activation of phase II enzymes, and secondarily on the inhibition of the carcinogen metabolism through the blocking of CYP enzymes. In addition to this, OL has been suggested to increase the nucleotide excision repair, which represents one of the major pathways for eliminating carcinogen DNA adducts; however, this effect has not been confirmed, as negative reports have also been published (O´Dwyer et al., 1997; Sparfel et al., 2002). With respect to GST, the mechanism by which OL enhances its level has been studied. Activation of such a cytoprotective enzyme seems to be related with a complex cellular signaling which includes the interaction of the Kelch ECH-associating protein (keap1) with the transcription factor NF-E2-related factor 2 (Nrf2), especially with the participation of the antioxidant response element-mediated regulation of Nrf2 (ARE) (Yates & Kensler, 2007). At this time, other dithiolethiones seem even more promising than OL in preventing cancer. One such example is the compound 3H-1,2-dithiole-3-thione, which has shown a potent induction of phase II enzymes, a powerful inhibition of AFB1-induced hepatic toxicity including the formation of hepatic preneoplastic lesions, and inhibition of hepatic AFB1- DNA adducts; besides, the chemical was found to cause significant increases of GST and/or NQO1 in 12 tissues in addition to the liver, and it is probably not an inducer of CYP

enzymes (Kensler et al., 1987; Roebuck et al., 2003; Zhang & Munday, 2008).

new dithiolethiones should be valuable.

**8. Vitamins** 

Future studies may include a structure-activity relationship among dithiolethiones, mainly to identify structural features that convey potent activation of Nrf2 and induction of phase II enzymes, the identification of mechanisms involved, as well as new biomarkers for evaluating their in vivo efficacy. Also, toxicity evaluations and clinical trials, especially with

These are organic compounds essential for the normal growth and development of a multicellular organism. A human fetus begins to develop from the nutrients it absorbs, including a certain amount of vitamins which facilitate the chemical reactions in different tissues. Vitamins have diverse biochemical functions such as the hormone-like regulation of mineral metabolism, regulation of cell and tissue growth as well as differentiation, besides acting as antioxidants, or precursors of enzyme cofactors. A number of efforts have been made to evaluate the useful impact of these compounds on the damaging effects induced by AFB1. Published studies have been made in in vitro and in vivo models, which include investigations in bacteria, cultured cells, experimental animals, or humans. The applied approaches go from the determination of their capacity to prevent various types of AFB1

al., 2006).

2003, 2009), its induction of embryo lethality in mouse (Garcia-Rodriguez et al., 2002), or its possible effect as a tumor promoter in rats (Nelson, 1992).

#### **7. Oltipraz**

Dithiolethiones are five-membered cyclic sulfur-containing compounds found in cruciferous vegetables, which have shown radioprotective, chemopreventive, chemotherapeutic, and antiviral activities (Kensler, 1997). In the context of our interest, a study made with lyophilized cabbage or cauliflower demonstrated a significant reduction in the rate of AFB1-induced carcinogenesis in rat (Boyd et al., 1982); subsequently, this chemopreventive effect has been thoroughly evaluated in regard to the drug oltipraz (OL), 5-(2-pirazinyl)-4-methyl-1,2-dithiole-3-thione (Figure 5). The mentioned dithiolethione was initially used because of its antischistosomal capacity in animals and humans, but later it was widely evaluated because of the detected potential to abolish or reduce the liver carcinogenesis induced by AFB1.

#### Fig. 5. Chemical structure of oltipraz

In an earlier study, rats were fed for 4 weeks with OL and gavaged with AFB1 during the second and third week of the assay (Kensler et al., 1987). In this report, the authors determined a significant reduction of focal areas with hepatocellular alterations achieved with the administration of OL, an effect shown by the staining of liver sections for gammaglutamyl transpeptidase. The research concluded that dietary concentrations of OL as low as 0.01 % powerfully inhibited the formation of presumptive liver preneoplastic lesions. Similarly, the ameliorating effect of OL on the AFB1- toxicity induced in rats was shown by a decrease in the mortality rate, in the levels of serum alanine amino transaminase and sorbitol dehydrogenase, and also because of the normal growing rate propitiated by OL during the aflatoxin treatment (Liu et al., 1988). A few years later, another study made in rats, clearly confirmed the OL protective effect on AFB1-hepatocarcinogenesis (Roebuck et al., 1991). In this report, 11 % of hepatocellular carcinoma and 9 % hepatocellular adenomas were observed in AFB1-treated, diet-fed rats, in contrast with no tumor development in OL treated animals. Moreover, rats in the OL group had a significantly longer life span and an increased survival free of liver tumors in comparison with animals under aflatoxin treatment; besides, the authors found at least 65% reduction in the liver aflatoxin-N7 guanine adducts in the OL-fed animals, a finding which suggested that the protection against hepatocarcinogenesis might have resulted from the marked decrease in this type of hepatic DNA adducts. Studies about the chemopreventive efficacy of OL were also made in other animals, such as the marmoset monkey and the tree shrew. These studies gave rise to variable, but positive results (Bammler et al., 2000; Li et al., 2000).

With respect to humans, several studies have been made to examine the chemopreventive effect of OL. Phase IIa and phase IIb clinical trials were performed in a rural township in China (Kensler et al., 1998b; Wang et al., 1999). These trials were randomized, placebocontrolled, double-blind studies of people who had ingested AFB1 via their usual diet. For the rationale of the study the authors considered that the AFB1 epoxides are substrates for glutation S transferase (GST), an enzyme which catalyzes the conjugation of the epoxide with reduced glutation, thus mitigating the formation of DNA adducts. In fact, the results showed an increase in the activity of GST related with a sustained low dose of OL (125 mg/day), also yielding a high level of the AFB1 urinary metabolite, mercapturic acid; however, an intermittent, high dose of OL inhibited the activation of phase I enzymes, as reflected by a lowering in the excretion of the metabolite AFBm1. Nevertheless, the results of another study made in the same population suggested that prevention of oxidative DNA damage by OL was not a relevant mechanism to explain its effect against AFB1 (Glintborg et al., 2006).

The above-mentioned reports, as well as others, have suggested that the major preventive action of OL is through the activation of phase II enzymes, and secondarily on the inhibition of the carcinogen metabolism through the blocking of CYP enzymes. In addition to this, OL has been suggested to increase the nucleotide excision repair, which represents one of the major pathways for eliminating carcinogen DNA adducts; however, this effect has not been confirmed, as negative reports have also been published (O´Dwyer et al., 1997; Sparfel et al., 2002). With respect to GST, the mechanism by which OL enhances its level has been studied. Activation of such a cytoprotective enzyme seems to be related with a complex cellular signaling which includes the interaction of the Kelch ECH-associating protein (keap1) with the transcription factor NF-E2-related factor 2 (Nrf2), especially with the participation of the antioxidant response element-mediated regulation of Nrf2 (ARE) (Yates & Kensler, 2007).

At this time, other dithiolethiones seem even more promising than OL in preventing cancer. One such example is the compound 3H-1,2-dithiole-3-thione, which has shown a potent induction of phase II enzymes, a powerful inhibition of AFB1-induced hepatic toxicity including the formation of hepatic preneoplastic lesions, and inhibition of hepatic AFB1- DNA adducts; besides, the chemical was found to cause significant increases of GST and/or NQO1 in 12 tissues in addition to the liver, and it is probably not an inducer of CYP enzymes (Kensler et al., 1987; Roebuck et al., 2003; Zhang & Munday, 2008).

Future studies may include a structure-activity relationship among dithiolethiones, mainly to identify structural features that convey potent activation of Nrf2 and induction of phase II enzymes, the identification of mechanisms involved, as well as new biomarkers for evaluating their in vivo efficacy. Also, toxicity evaluations and clinical trials, especially with new dithiolethiones should be valuable.

#### **8. Vitamins**

264 Aflatoxins – Detection, Measurement And Control

2003, 2009), its induction of embryo lethality in mouse (Garcia-Rodriguez et al., 2002), or its

Dithiolethiones are five-membered cyclic sulfur-containing compounds found in cruciferous vegetables, which have shown radioprotective, chemopreventive, chemotherapeutic, and antiviral activities (Kensler, 1997). In the context of our interest, a study made with lyophilized cabbage or cauliflower demonstrated a significant reduction in the rate of AFB1-induced carcinogenesis in rat (Boyd et al., 1982); subsequently, this chemopreventive effect has been thoroughly evaluated in regard to the drug oltipraz (OL), 5-(2-pirazinyl)-4-methyl-1,2-dithiole-3-thione (Figure 5). The mentioned dithiolethione was initially used because of its antischistosomal capacity in animals and humans, but later it was widely evaluated because of

In an earlier study, rats were fed for 4 weeks with OL and gavaged with AFB1 during the second and third week of the assay (Kensler et al., 1987). In this report, the authors determined a significant reduction of focal areas with hepatocellular alterations achieved with the administration of OL, an effect shown by the staining of liver sections for gammaglutamyl transpeptidase. The research concluded that dietary concentrations of OL as low as 0.01 % powerfully inhibited the formation of presumptive liver preneoplastic lesions. Similarly, the ameliorating effect of OL on the AFB1- toxicity induced in rats was shown by a decrease in the mortality rate, in the levels of serum alanine amino transaminase and sorbitol dehydrogenase, and also because of the normal growing rate propitiated by OL during the aflatoxin treatment (Liu et al., 1988). A few years later, another study made in rats, clearly confirmed the OL protective effect on AFB1-hepatocarcinogenesis (Roebuck et al., 1991). In this report, 11 % of hepatocellular carcinoma and 9 % hepatocellular adenomas were observed in AFB1-treated, diet-fed rats, in contrast with no tumor development in OL treated animals. Moreover, rats in the OL group had a significantly longer life span and an increased survival free of liver tumors in comparison with animals under aflatoxin treatment; besides, the authors found at least 65% reduction in the liver aflatoxin-N7 guanine adducts in the OL-fed animals, a finding which suggested that the protection against hepatocarcinogenesis might have resulted from the marked decrease in this type of hepatic DNA adducts. Studies about the chemopreventive efficacy of OL were also made in other animals, such as the marmoset monkey and the tree shrew. These studies gave rise to

With respect to humans, several studies have been made to examine the chemopreventive effect of OL. Phase IIa and phase IIb clinical trials were performed in a rural township in China (Kensler et al., 1998b; Wang et al., 1999). These trials were randomized, placebo-

variable, but positive results (Bammler et al., 2000; Li et al., 2000).

the detected potential to abolish or reduce the liver carcinogenesis induced by AFB1.

possible effect as a tumor promoter in rats (Nelson, 1992).

Fig. 5. Chemical structure of oltipraz

**7. Oltipraz** 

These are organic compounds essential for the normal growth and development of a multicellular organism. A human fetus begins to develop from the nutrients it absorbs, including a certain amount of vitamins which facilitate the chemical reactions in different tissues. Vitamins have diverse biochemical functions such as the hormone-like regulation of mineral metabolism, regulation of cell and tissue growth as well as differentiation, besides acting as antioxidants, or precursors of enzyme cofactors. A number of efforts have been made to evaluate the useful impact of these compounds on the damaging effects induced by AFB1. Published studies have been made in in vitro and in vivo models, which include investigations in bacteria, cultured cells, experimental animals, or humans. The applied approaches go from the determination of their capacity to prevent various types of AFB1

Aflatoxin B1 - Prevention of Its Genetic Damage by Means of Chemical Agents 267

devoid of the vitamin developed liver cancer, contrary to few cases in rats which received it (Nyandieka & Wakhisi, 1993). However, a study aimed at determining the vitamin inhibitory effect on liver preneoplastic foci showed negative results, a finding which was probably related with an excess of the vitamin in the assay; this, nonetheless, helped to stress the relevance of selecting appropriate experimental conditions in chemoprevention

Finally, although there is a deplorable scarcity of studies in humans, AFB1-albumin adducts were quantified in a high risk Ghanaian population, where a relationship was determined between a high mycotoxin level with decreased levels of the vitamins A and E, suggesting then, that such deficiency may significantly influence the incidence of adverse health effects

Vitamin C and E are other compounds tested against the genotoxic damage induced by AFB1. With respect to these chemicals, a study made by means of the *Salmonella typhimurium* test (strains TA98 and TA100) showed that although both vitamins prevented the expression of AFB1-induced mutagenesis, vitamin E was more potent, and also that its effect was related with the metabolism of the mycotoxin, whereas vitamin C was involved in both the metabolic and post-metabolic levels of the AFB1 mutagenesis assay (Raina & Gurtoo, 1985). This result was congruent with the protective, dose-dependent effect determined for both vitamins against the SCE induced by AFB1 in cultured human lymphocytes (Alpsoy et al., 2009). In this study, the order of protective efficacy was vitamin C-vitamin E-vitamin A. In regard to vitamin E, however, the indicated positive results were contrary to those reported by Karekar et al. (2000) who applied two short term genotoxicity assays ─ the Ames test and the Drosophila wing spot test ─ and they found no antimutagenic response of the vitamin; moreover, woodchuck hepatocytes that were treated with four doses of [3H]AFB1 or with different combinations of the toxin and vitamins C and E for 6 h resulted in an effect of vitamin C for inhibiting AFB1-DNA binding; contrarily, an enhancement of covalent binding of AFB1 to DNA by vitamin E was observed (Yu et al., 1994). Also, negative results were found when evaluating the protection of such vitamin in SCE induced by AFB1 in V79 cells (Deng et al., 1988). These results clearly suggest the need for further research to understand the complex role of these vitamins in the mutagenesis and carcinogenesis of the aflatoxin. Such a complex response was also reported in rats fed on a variable diet of vitamin E (Cassand et al., 1993). Animals on a diet supplemented with a low amount of the vitamin (0.5 IU) increased P-450 IIB and IIIA enzyme activity, whereas a higher vitamin supplemented diet (5 IU) reduced these specific activities. However, lipid peroxidation was increased in the vitamin E free diet animals and strongly decreased in the supplemented group. Nevertheless, in a subsequent study (Karakilcik et al., 2004) a significant increase was found in the level of various liver enzymes in rabbits fed a diet with AFB1, while such activities were lower in the groups receiving the mycotoxin plus vitamins C or E, whether alone or combined. In spite of these controversial reports on antigenotoxicity, another study made in rats to determine the preventive capacity of vitamins C and E on the development of liver cancer gave strong positive results, because only few animals under vitamin treatment suffered the illness along the 24 months of the assay (Nyandieka &

On the other hand, some studies aimed to test the effect of specific types of vitamin B have given inconclusive results. In the case of riboflavin, an earlier assay using the *Salmonella typhimurium,* strain T100, rat-liver microsome system, concluded that with lower

studies (Gradelet et al., 1998).

(Tang et al., 2009).

Wakhisi, 1993).

genotoxic alterations, including adduction, to their modulatory effects on the AFB1 metabolism, or their participation as anticarcinogenic agents.

In regard to vitamin A, initial reports about its protective capacity were made by means of the Ames *Salmonella*/mammalian microsomes test. Such studies showed a relevant concentration-dependent decrease of the mutagenicity induced by AFB1. The effects were determined in strains TA98 and TA100, where the authors considered that the observed capacity of the vitamin could be related with the inhibition of AFB1 metabolism or with an increased breakdown of the active metabolite (Busk & Ahlborg, 1980; Raina & Gurtoo, 1985). Other strains (TA102 and TA1535) were also tested and revealed positive results (Qin & Huang, 1986). In Chinese hamster V79 cells, (Huang et al., 1982) found a similar effect. In this assay, dose and time dependent inhibition of AFB1-induced SCE, as well as correction of the cell cycle delay produced by the toxin was achieved by adding vitamin A to the cultures. Moreover, Qin et al. (1985) confirmed the indicated finding and extended it to determine a similar effect of the vitamin over the amount of chromosomal aberrations induced by AFB1. A few years later, S9 fractions obtained from mice with a high vitamin A liver level were found to be less potent in activating AFB1 than those with a low liver level; also, the first ones proved to be related with a stronger reduction of SCE in mice administered aflatoxin with respect to the effect in mice with a low vitamin A level, which therefore confirmed the role of such vitamin to ameliorate the genotoxic damage (Quin & Huang, 1985). In this period, Suphakarn et al. (1983) also determined an enhancement of liver and colon cancer in rats with a vitamin A deficient diet and exposed to AFB1. The authors evaluated factors such as liver morphology, enterohepatic recirculation, level of reduced glutathione in liver, and conjugating capacity to GST, and they suggested that their results may have been related with the influence of the vitamin on the binding of AFB1 to cellular macromolecules, partially influenced through enzymatic mechanisms. With the purpose of learning more about the preventive effect of vitamin A, Webster et al. (1996) applied the approach of modulating its ingested amount. They found that rats with a deficiency of vitamin showed a high level of DNA single strand breaks induced by AFB1, as well as a decrease in various repair enzymes subsequent to DNA damage, although correction of these two parameters was achieved with vitamin supplementation. In regard to the capacity of AFB1 for inducing DNA adducts, an in vitro assay using a microsome catalyzed reaction showed that the addition of vitamin A to the system produced a dose-dependent inhibition of the adduction (Firozi et al., 1987). Similar results were found studying woodchuck hepatocytes (Yu et al., 1994).

The information indicated above suggested to researchers that the main action of vitamin A (as well as of other vitamins) was on the initiating step of AFB1 carcinogenesis, yet there still remained studies to be done so as to clarify the issue on the preventive biochemical action of the vitamin (Bhattacharya et al., 1989; Decoudu et al., 1992).

The antigenotoxic and antitoxic potential of vitamin A was determined in experimental mice. In these animals, a decrease in the toxin-induced clastogenicity in both mitotic and meiotic chromosomes was reported, as well as inhibition in sperm abnormalities (Sinha & Darmshila, 1994). Besides, the antigenotoxic effect of vitamin A was also found in human lymphocytes (Alpsoy et al., 2009); in these cells the authors reported a significant, dosedependent reduction of the SCE induced by 5 uM of AFB1, with the lowest protective concentration being 0.5 uM.

With respect to the anticarcinogenic potential, a report established such an effect in a 2-year follow up of AFB1-administered rats where it was observed that most animals fed a diet

genotoxic alterations, including adduction, to their modulatory effects on the AFB1

In regard to vitamin A, initial reports about its protective capacity were made by means of the Ames *Salmonella*/mammalian microsomes test. Such studies showed a relevant concentration-dependent decrease of the mutagenicity induced by AFB1. The effects were determined in strains TA98 and TA100, where the authors considered that the observed capacity of the vitamin could be related with the inhibition of AFB1 metabolism or with an increased breakdown of the active metabolite (Busk & Ahlborg, 1980; Raina & Gurtoo, 1985). Other strains (TA102 and TA1535) were also tested and revealed positive results (Qin & Huang, 1986). In Chinese hamster V79 cells, (Huang et al., 1982) found a similar effect. In this assay, dose and time dependent inhibition of AFB1-induced SCE, as well as correction of the cell cycle delay produced by the toxin was achieved by adding vitamin A to the cultures. Moreover, Qin et al. (1985) confirmed the indicated finding and extended it to determine a similar effect of the vitamin over the amount of chromosomal aberrations induced by AFB1. A few years later, S9 fractions obtained from mice with a high vitamin A liver level were found to be less potent in activating AFB1 than those with a low liver level; also, the first ones proved to be related with a stronger reduction of SCE in mice administered aflatoxin with respect to the effect in mice with a low vitamin A level, which therefore confirmed the role of such vitamin to ameliorate the genotoxic damage (Quin & Huang, 1985). In this period, Suphakarn et al. (1983) also determined an enhancement of liver and colon cancer in rats with a vitamin A deficient diet and exposed to AFB1. The authors evaluated factors such as liver morphology, enterohepatic recirculation, level of reduced glutathione in liver, and conjugating capacity to GST, and they suggested that their results may have been related with the influence of the vitamin on the binding of AFB1 to cellular macromolecules, partially influenced through enzymatic mechanisms. With the purpose of learning more about the preventive effect of vitamin A, Webster et al. (1996) applied the approach of modulating its ingested amount. They found that rats with a deficiency of vitamin showed a high level of DNA single strand breaks induced by AFB1, as well as a decrease in various repair enzymes subsequent to DNA damage, although correction of these two parameters was achieved with vitamin supplementation. In regard to the capacity of AFB1 for inducing DNA adducts, an in vitro assay using a microsome catalyzed reaction showed that the addition of vitamin A to the system produced a dose-dependent inhibition of the adduction (Firozi et al., 1987). Similar results

metabolism, or their participation as anticarcinogenic agents.

were found studying woodchuck hepatocytes (Yu et al., 1994).

the vitamin (Bhattacharya et al., 1989; Decoudu et al., 1992).

concentration being 0.5 uM.

The information indicated above suggested to researchers that the main action of vitamin A (as well as of other vitamins) was on the initiating step of AFB1 carcinogenesis, yet there still remained studies to be done so as to clarify the issue on the preventive biochemical action of

The antigenotoxic and antitoxic potential of vitamin A was determined in experimental mice. In these animals, a decrease in the toxin-induced clastogenicity in both mitotic and meiotic chromosomes was reported, as well as inhibition in sperm abnormalities (Sinha & Darmshila, 1994). Besides, the antigenotoxic effect of vitamin A was also found in human lymphocytes (Alpsoy et al., 2009); in these cells the authors reported a significant, dosedependent reduction of the SCE induced by 5 uM of AFB1, with the lowest protective

With respect to the anticarcinogenic potential, a report established such an effect in a 2-year follow up of AFB1-administered rats where it was observed that most animals fed a diet devoid of the vitamin developed liver cancer, contrary to few cases in rats which received it (Nyandieka & Wakhisi, 1993). However, a study aimed at determining the vitamin inhibitory effect on liver preneoplastic foci showed negative results, a finding which was probably related with an excess of the vitamin in the assay; this, nonetheless, helped to stress the relevance of selecting appropriate experimental conditions in chemoprevention studies (Gradelet et al., 1998).

Finally, although there is a deplorable scarcity of studies in humans, AFB1-albumin adducts were quantified in a high risk Ghanaian population, where a relationship was determined between a high mycotoxin level with decreased levels of the vitamins A and E, suggesting then, that such deficiency may significantly influence the incidence of adverse health effects (Tang et al., 2009).

Vitamin C and E are other compounds tested against the genotoxic damage induced by AFB1. With respect to these chemicals, a study made by means of the *Salmonella typhimurium* test (strains TA98 and TA100) showed that although both vitamins prevented the expression of AFB1-induced mutagenesis, vitamin E was more potent, and also that its effect was related with the metabolism of the mycotoxin, whereas vitamin C was involved in both the metabolic and post-metabolic levels of the AFB1 mutagenesis assay (Raina & Gurtoo, 1985). This result was congruent with the protective, dose-dependent effect determined for both vitamins against the SCE induced by AFB1 in cultured human lymphocytes (Alpsoy et al., 2009). In this study, the order of protective efficacy was vitamin C-vitamin E-vitamin A. In regard to vitamin E, however, the indicated positive results were contrary to those reported by Karekar et al. (2000) who applied two short term genotoxicity assays ─ the Ames test and the Drosophila wing spot test ─ and they found no antimutagenic response of the vitamin; moreover, woodchuck hepatocytes that were treated with four doses of [3H]AFB1 or with different combinations of the toxin and vitamins C and E for 6 h resulted in an effect of vitamin C for inhibiting AFB1-DNA binding; contrarily, an enhancement of covalent binding of AFB1 to DNA by vitamin E was observed (Yu et al., 1994). Also, negative results were found when evaluating the protection of such vitamin in SCE induced by AFB1 in V79 cells (Deng et al., 1988). These results clearly suggest the need for further research to understand the complex role of these vitamins in the mutagenesis and carcinogenesis of the aflatoxin. Such a complex response was also reported in rats fed on a variable diet of vitamin E (Cassand et al., 1993). Animals on a diet supplemented with a low amount of the vitamin (0.5 IU) increased P-450 IIB and IIIA enzyme activity, whereas a higher vitamin supplemented diet (5 IU) reduced these specific activities. However, lipid peroxidation was increased in the vitamin E free diet animals and strongly decreased in the supplemented group. Nevertheless, in a subsequent study (Karakilcik et al., 2004) a significant increase was found in the level of various liver enzymes in rabbits fed a diet with AFB1, while such activities were lower in the groups receiving the mycotoxin plus vitamins C or E, whether alone or combined. In spite of these controversial reports on antigenotoxicity, another study made in rats to determine the preventive capacity of vitamins C and E on the development of liver cancer gave strong positive results, because only few animals under vitamin treatment suffered the illness along the 24 months of the assay (Nyandieka & Wakhisi, 1993).

On the other hand, some studies aimed to test the effect of specific types of vitamin B have given inconclusive results. In the case of riboflavin, an earlier assay using the *Salmonella typhimurium,* strain T100, rat-liver microsome system, concluded that with lower

Aflatoxin B1 - Prevention of Its Genetic Damage by Means of Chemical Agents 269

The cell wall of yeast, as well as that of other microorganisms is composed of polyssacharides, mainly mannans, glucans, and glucomannans, some of which have been studied for their AFB1 protective effect. Mannan is a highly branched olygossacharide constituted by a main chain of -(1,6)-D-mannoses linearly attached, and with -(1,2) and -(1,3)-D-mannose branches. In a first report mice were fed AFB1-contaminated corn, and AFB1 treated grain plus three doses of mannan (including the appropriate control groups). The assay lasted four weeks and the measurements included, weight, micronuclei, cytotoxicity index, and SCE (Madrigal-Santillán et al., 2007). Results showed that mice fed AFB1 had a significant weight decrease, as well as a significant increase in the rate of MNNE and SCE, while animals fed the combined regime presented a 25 % weight increase with respect to animals treated with AFB1 alone, as well as a reduction in the level of MNNE and SCE (about 70 % with the high two doses). In a subsequent report, the authors confirmed the protective effect of mannan in mouse hepatocytes which were analyzed with the comet assay at 4, 10 and 16 h of exposure (Madrigal-Santillán et al., 2009). In such study, the best preventive effect of mannan was found at 10 h with the high tested dose (700 mg/kg). Moreover, the authors proposed a supramolecular complex between mannan and the aflatoxin based on the melting points, and the UV spectra of the crystals from the

Glucans are a heterogeneous group of glucose polymers, consisting of a backbone of β(1,3) linked β-D-glucopyranosyl units with β(1,6)-linked side chains of varying distribution and length (Akramiene et al., 2007). Besides its immunostimulant effect, the compound has been reported to have chemoprotective potential against a number of mutagenic agents (Akramiene et al., 2007; Mantovani et al., 2007); also, interaction of glucans with AFB1 including the participation of hydroxyl, ketone, and lactone groups was proposed as the basis for the formation of hydrogen bonds and van der Waals interactions (Yiannikouris et al., 2006). However, in spite of such information, very few studies have been made in regard to the antigenotoxic potential of glucans on the damage induced by AFB1. An investigation similar to the described above by Madrigal-Santillán et al. (2009) but testing the antigenotoxicity of glucan and glucomannan in mice hepatocytes showed a positive effect for the two agents (Madrigal-Santillán, 2004). DNA damage was quantified by means of the comet assay at 4, 10 at 16 h after the chemicals exposure. Glucan showed a protective effect with the two low doses tested (400, and 700 mg/kg), reaching about 40% as the highest reduction of the damage induced by AFB1; glucomannan, however, showed a significant response with all the three tested doses, reaching an inhibition as high as 80 % at 10 h of

Our purpose in this section is not to show an extensive list of the agents tested against the genotoxicity of AFB1, but rather the variability of such agents, which goes from single compounds to mixtures with different complexity. These investigations may be motivated by the mutagenic potency of AFB1, which make it a relevant candidate for demonstrating the capacity of antimutagens, as well as by the need for finding efficient agents to prevent

Ellagic acid and a phenolic extract obtained from the bean (*Phaseolus vulgaris*) are examples of phenolic compounds studied for their use in controlling the mutagenicity of AFB1. In both cases *Salmonella typhimurium* (strain TA98 and TA100) was used as the test model, and the

independent compounds and a co-crystalization of both chemicals.

treatment.

**10. Miscellaneous agents** 

the serious damage that such mutagen can provoke.

concentrations of AFB1 the effect of the vitamin was very strong (Bhattacharya et al., 1987). However, another in vitro and in vivo study suggested a variable role of the compound with respect to the AFB1 metabolic activation, an effect which was related with the tested amount of the vitamin (Prabhu et al., 1989). Then, in a subsequent report made in rats under riboflavin supplementation, a clear, positive effect was determined on the DNA damage induced by AFB1 by quantifying the reversion of DNA single strand breaks (Webster et al., 1996). Folic acid has also been evaluated. In this case, a survey made in high risk Chinese individuals concluded that increased folate levels may be inversely associated with the development of liver damage and hepatocellular carcinoma (Welzel et al., 2007).

#### **9. Probiotics and microbial cell wall components**

In the context of the exposed theme, biological decontamination seems attractive because it works under mild, environmentally friendly conditions. The AFB1 detoxification potential of probiotics such as yeast and lactic acid bacteria, among other microorganisms, has been evaluated in light of their adsorbent capacity that prevents the transfer of aflatoxin to the intestinal tract of humans and animals (Wu et al., 2009). This effect has been reported for various species of *Lactobacillus,* including *L. casei, L. plantarum, L. fermentum, and L. rhamnosus*; moreover, the participation of teichoic acids has been suggested to play a key role in the binding ability of some species toward AFB1 (Fazeli et al., 2009; Gratz et al., 2007; Hernandez-Mendoza et al., 2009). Although the antimutagenic capacity of fermented foods and probiotics is known, few studies in respect to aflatoxin have been done with bacteria; an example is the report made in Caco-2cells treated with *Lactobacillus rhamnosus* strain GG, which showed protection against AFB1-induced reductions in transephitelial resistance, as well as reductions in DNA fragmentations assessed by extracting DNA and separating intact and damaged DNA by the use of gel electrophoresis (Grats et al., 2007). In regard to probiotics, there is an interesting study made on ninety healthy young men from Guangzhou, China whose diet was supplemented with a probiotic mixture that induced a reduction of the biologically effective dose of aflatoxin exposure, suggesting an effective dietary approach to decrease the risk of liver cancer. This conclusion was reached after quantifying the urinary excretion of AFB1-N7-guanine in the evaluated population (El-Nezami et al., 2006).

Research on the yeast *Saccharomyces cereviciae* (Sc) has confirmed its decontaminating ability through its binding with AFB1, which may depend on the used strain and other experimental conditions (Shetty et al., 2007). Besides, the potential of Sc to ameliorate the effects of aflatoxicosis was clearly established in broiler chicks or Japanese quail by evaluating a number of biochemical and organic parameters (Parlat et al., 2001; Stanley et al., 1993). The above-mentioned information is congruent with the antigenotoxic effect observed in mice fed with AFB1 contaminated corn (Madrigal-Santillán et al., 2006). In this study, the animals were experimentally fed with the tested chemical for six weeks; the results observed in the groups treated with the yeast showed a significant improvement in the weight loss induced by AFB1, and a decrease of more than 60 % in the level of micronuclei induced by the toxin in normochromatic erythrocytes, as well as a similar reduction in the level of SCE in mouse bone marrow cells, effects that were related with the adsorbing capacity of the yeast. Besides, the study revealed a recovery to normal parameters in about three weeks without the aflatoxin administration, which suggest the usefulness of periodical monitoring of commodities at risk.

concentrations of AFB1 the effect of the vitamin was very strong (Bhattacharya et al., 1987). However, another in vitro and in vivo study suggested a variable role of the compound with respect to the AFB1 metabolic activation, an effect which was related with the tested amount of the vitamin (Prabhu et al., 1989). Then, in a subsequent report made in rats under riboflavin supplementation, a clear, positive effect was determined on the DNA damage induced by AFB1 by quantifying the reversion of DNA single strand breaks (Webster et al., 1996). Folic acid has also been evaluated. In this case, a survey made in high risk Chinese individuals concluded that increased folate levels may be inversely associated with the

In the context of the exposed theme, biological decontamination seems attractive because it works under mild, environmentally friendly conditions. The AFB1 detoxification potential of probiotics such as yeast and lactic acid bacteria, among other microorganisms, has been evaluated in light of their adsorbent capacity that prevents the transfer of aflatoxin to the intestinal tract of humans and animals (Wu et al., 2009). This effect has been reported for various species of *Lactobacillus,* including *L. casei, L. plantarum, L. fermentum, and L. rhamnosus*; moreover, the participation of teichoic acids has been suggested to play a key role in the binding ability of some species toward AFB1 (Fazeli et al., 2009; Gratz et al., 2007; Hernandez-Mendoza et al., 2009). Although the antimutagenic capacity of fermented foods and probiotics is known, few studies in respect to aflatoxin have been done with bacteria; an example is the report made in Caco-2cells treated with *Lactobacillus rhamnosus* strain GG, which showed protection against AFB1-induced reductions in transephitelial resistance, as well as reductions in DNA fragmentations assessed by extracting DNA and separating intact and damaged DNA by the use of gel electrophoresis (Grats et al., 2007). In regard to probiotics, there is an interesting study made on ninety healthy young men from Guangzhou, China whose diet was supplemented with a probiotic mixture that induced a reduction of the biologically effective dose of aflatoxin exposure, suggesting an effective dietary approach to decrease the risk of liver cancer. This conclusion was reached after quantifying the urinary excretion of AFB1-N7-guanine in the evaluated population (El-

Research on the yeast *Saccharomyces cereviciae* (Sc) has confirmed its decontaminating ability through its binding with AFB1, which may depend on the used strain and other experimental conditions (Shetty et al., 2007). Besides, the potential of Sc to ameliorate the effects of aflatoxicosis was clearly established in broiler chicks or Japanese quail by evaluating a number of biochemical and organic parameters (Parlat et al., 2001; Stanley et al., 1993). The above-mentioned information is congruent with the antigenotoxic effect observed in mice fed with AFB1 contaminated corn (Madrigal-Santillán et al., 2006). In this study, the animals were experimentally fed with the tested chemical for six weeks; the results observed in the groups treated with the yeast showed a significant improvement in the weight loss induced by AFB1, and a decrease of more than 60 % in the level of micronuclei induced by the toxin in normochromatic erythrocytes, as well as a similar reduction in the level of SCE in mouse bone marrow cells, effects that were related with the adsorbing capacity of the yeast. Besides, the study revealed a recovery to normal parameters in about three weeks without the aflatoxin administration, which suggest the usefulness of

development of liver damage and hepatocellular carcinoma (Welzel et al., 2007).

**9. Probiotics and microbial cell wall components** 

Nezami et al., 2006).

periodical monitoring of commodities at risk.

The cell wall of yeast, as well as that of other microorganisms is composed of polyssacharides, mainly mannans, glucans, and glucomannans, some of which have been studied for their AFB1 protective effect. Mannan is a highly branched olygossacharide constituted by a main chain of -(1,6)-D-mannoses linearly attached, and with -(1,2) and -(1,3)-D-mannose branches. In a first report mice were fed AFB1-contaminated corn, and AFB1 treated grain plus three doses of mannan (including the appropriate control groups). The assay lasted four weeks and the measurements included, weight, micronuclei, cytotoxicity index, and SCE (Madrigal-Santillán et al., 2007). Results showed that mice fed AFB1 had a significant weight decrease, as well as a significant increase in the rate of MNNE and SCE, while animals fed the combined regime presented a 25 % weight increase with respect to animals treated with AFB1 alone, as well as a reduction in the level of MNNE and SCE (about 70 % with the high two doses). In a subsequent report, the authors confirmed the protective effect of mannan in mouse hepatocytes which were analyzed with the comet assay at 4, 10 and 16 h of exposure (Madrigal-Santillán et al., 2009). In such study, the best preventive effect of mannan was found at 10 h with the high tested dose (700 mg/kg). Moreover, the authors proposed a supramolecular complex between mannan and the aflatoxin based on the melting points, and the UV spectra of the crystals from the independent compounds and a co-crystalization of both chemicals.

Glucans are a heterogeneous group of glucose polymers, consisting of a backbone of β(1,3) linked β-D-glucopyranosyl units with β(1,6)-linked side chains of varying distribution and length (Akramiene et al., 2007). Besides its immunostimulant effect, the compound has been reported to have chemoprotective potential against a number of mutagenic agents (Akramiene et al., 2007; Mantovani et al., 2007); also, interaction of glucans with AFB1 including the participation of hydroxyl, ketone, and lactone groups was proposed as the basis for the formation of hydrogen bonds and van der Waals interactions (Yiannikouris et al., 2006). However, in spite of such information, very few studies have been made in regard to the antigenotoxic potential of glucans on the damage induced by AFB1. An investigation similar to the described above by Madrigal-Santillán et al. (2009) but testing the antigenotoxicity of glucan and glucomannan in mice hepatocytes showed a positive effect for the two agents (Madrigal-Santillán, 2004). DNA damage was quantified by means of the comet assay at 4, 10 at 16 h after the chemicals exposure. Glucan showed a protective effect with the two low doses tested (400, and 700 mg/kg), reaching about 40% as the highest reduction of the damage induced by AFB1; glucomannan, however, showed a significant response with all the three tested doses, reaching an inhibition as high as 80 % at 10 h of treatment.

#### **10. Miscellaneous agents**

Our purpose in this section is not to show an extensive list of the agents tested against the genotoxicity of AFB1, but rather the variability of such agents, which goes from single compounds to mixtures with different complexity. These investigations may be motivated by the mutagenic potency of AFB1, which make it a relevant candidate for demonstrating the capacity of antimutagens, as well as by the need for finding efficient agents to prevent the serious damage that such mutagen can provoke.

Ellagic acid and a phenolic extract obtained from the bean (*Phaseolus vulgaris*) are examples of phenolic compounds studied for their use in controlling the mutagenicity of AFB1. In both cases *Salmonella typhimurium* (strain TA98 and TA100) was used as the test model, and the

Aflatoxin B1 - Prevention of Its Genetic Damage by Means of Chemical Agents 271

In light of the serious effects that AFB1 contamination can originate, the authors agree that different socio-economical and toxicological approaches should be carried out for its elimination or control, including specific strategies regarding regulatory, supervisory, educational, scientific and technologic issues. Basic knowledge on the metabolism and the molecular and cellular fate of AFB1 is presently known, and various models have been used to test the effects of a number of chemopreventive agents, some of which have shown promising results, suggesting then, the pertinence of continuing with such strategy. However, it is reasonable to have a deeper knowledge on the chemical characteristics of each AFB1 metabolite, as well as on their interactions with macromolecules and cells, and to identify the more sensitive biomarkers for the assayed damage; this will be of help in designing more appropriate experimental projects, or clinical trials with the best candidates detected, in addition to preventing the selected genotoxic damage with more efficacy. At present, only a few agents have been tested in humans for evaluating their capacity of protection against AFB1 damage, although numerous chemicals have been evaluated in an almost isolated experimental form and have presented favorable results; therefore, extensive studies on these agents should be carried out so as to gain knowledge on their safety, efficacy, and mechanism of action, in order to select those more suitable for

Abraham, S. (1991). Inhibitory effects of coffee on the genotoxicity of carcinogens in mice. *Mutation Research*. Vol. 262, No. 2 (February 1991), pp. 109-114, ISSN 0027-5107 Akramienė, D., Kondrotas, A., Didžiapetrienė, J., & Kėvelaitis, E. (2007). Effects of B-glucans

Alpsoy, L., Agar, G., & Ikbal, M. (2009). Protective role of vitamins A, C, and E against the

*and Industrial Health,* Vol. 25, No. 3, (April 2009), pp. 183-188, ISSN 0748-2337 Ames, B., Durston, W., Yamasaki, E., & Lee, F. (1973) Carcinogens are Mutagens: A Simple

*America*, Vol. 70, No. 8, (August 1973), pp. 2281-2285, ISSN 0027-8424 Ames, B. (1983) Dietary carcinogens and anticarcinogens. Oxygen radicals and degenerative

Anguiano-Ruvalcaba, G., Vargas-Cortina, A., & Guzmán-De Peña, D. (2005). Inactivación de

Anwar, W., Khalil, M., & Wild, C. (1994). Micronuclei, chromosomal aberrations and

*Mutation Research* Vol. 322, No. 1, (July 1994), pp. 61–67, ISSN 0027-5107.

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genotoxic damage induced by aflatoxin B1 in cultured human lymphocytes. *Toxicology* 

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diseases. *Science*, Vol. 221, No. 221, (September 1983), pp. 1256-1264, ISSN 0036-

aflatoxina B1 y aflatoxicol por nixtamalización tradicional del maíz y su regeneración por acidificación de la masa. *Salud Pública de México*. Vol. 47, No. 5,

aflatoxin albumin adducts in experimental animals after exposure to aflatoxin B1.

**11. Conclusions** 

chemopreventive purposes.

1010-660X

8075

(2005), pp. 369-375

**12. References** 

obtained results showed a concentration-dependent antimutagenic effect, which was more clearly expressed when the compounds were tested at the same time. The authors suggested the formation of a chemical complex between the involved agents as an explanation for the protective effect (Loarca-Piña et al., 1998; Cardador-Martinez et al., 2002).

Green and black teas are known as efficient antimutagenic and anticarcinogenic agents. In the case of AFB1, tea polyphenols from both teas were tested against its mutagenicity by means of the *Salmonella typhimurium* assay (strain TA98). In the report the authors determined a sharp decrease toward the mutagenic effect of the aflatoxin (Weisburger et al., 1996). Besides, results obtained in rat bone marrow cells treated with AFB1 in vivo revealed that the administration of green tea 24 h before administering the mutagen produced a significant reduction in the number of structural chromosomal aberrations (Ito et al., 1989). A confirmation of the green tea protective effect was determined in 352 human blood and urine samples that corresponded to a 3-month trial of individuals under green tea consumption (Tang et al., 2008). The authors measured AFB1-albumin adducts, AFBM1, and AFB1-mercapturic acid, and concluded that green tea effectively modulates the metabolism and metabolic activation of AFB1.

A number of plant flavonoids were tested against the effect of AFB1 by means of the *Salmonella typhimurium* assay (TA98 and TA100 strains), and some of them showed an efficient antimutagenic capacity: kaempferol, morin, fisetin, biochanin A, and rutin (Francis et al., 1989). Also, it was reported that kolaviron, a flavonoid from the seeds of *Garcinia kola* was able to inhibit the amount of micronuclei and the hepatic oxidative damage induced by AFB1 in rats (Farombi et al., 2005).

The determination of ammonia as antimutagen is included in this review considering that the chemical has been used as one of the agents to decontaminate AFB1; therefore, confirmation of its utility through genetic endpoints seems interesting. In the described report, mice were fed for four weeks with AFB1 contaminated corn and concomitantly treated with ammonium hydroxide (Marquez-Marquez et al., 1993). The results showed a significant reduction in the rate of micronucleated normochromatic erythrocytes starting from the first week of the assay, and at the fourth week of treatment the inhibition reached 60 %; besides, at the last week of the test, the quantification of SCE showed an inhibition of 55 % in comparison with the level determined in the AFB1 treated group.

Coffee is a beverage of habitual consumption that has shown controversial results concerning its genotoxic/antigenotoxic potential; however, there is an interesting study by Abraham (1991) who evaluated the inhibitory effect of standard instant coffee on the number of mice bone marrow micronuclei. Mice were orally administered coffee 2 and 20 h before injecting the carcinogen, and observations made at 28 and 48 h showed a dosedependent decrease in the rate of micronuclei, with a reduction of more than 60 % with the high tested dose (500 mg/kg).

In regard to constituents of apiaceous vegetables, such as carrots, parsnips, celery or parsley, Peterson et al. (2006) used a methoxyresorufin O-demethylase assay and a trp-recombination assay in *Saccharomyces cereviciae*, and found that 5-methoxypsoralen, and 8-methoxypsoralen reduced the CYP1A2-mediated mutagenesis of AFB1. In the same context, it was reported the hepatoprotective effect of ethanolic extract of *Phyllantus amarus* on AFB1-induced damage in mice, as well as the protective effect of soybean saponins against the aflatoxin in the *Salmonella typhimurium* assay, and a significant decrease of DNAadduct formation in human liver hepatoma cells (Jun et al., 2002; Naaz et al., 2007).

#### **11. Conclusions**

270 Aflatoxins – Detection, Measurement And Control

obtained results showed a concentration-dependent antimutagenic effect, which was more clearly expressed when the compounds were tested at the same time. The authors suggested the formation of a chemical complex between the involved agents as an explanation for the

Green and black teas are known as efficient antimutagenic and anticarcinogenic agents. In the case of AFB1, tea polyphenols from both teas were tested against its mutagenicity by means of the *Salmonella typhimurium* assay (strain TA98). In the report the authors determined a sharp decrease toward the mutagenic effect of the aflatoxin (Weisburger et al., 1996). Besides, results obtained in rat bone marrow cells treated with AFB1 in vivo revealed that the administration of green tea 24 h before administering the mutagen produced a significant reduction in the number of structural chromosomal aberrations (Ito et al., 1989). A confirmation of the green tea protective effect was determined in 352 human blood and urine samples that corresponded to a 3-month trial of individuals under green tea consumption (Tang et al., 2008). The authors measured AFB1-albumin adducts, AFBM1, and AFB1-mercapturic acid, and concluded that green tea effectively modulates the metabolism

A number of plant flavonoids were tested against the effect of AFB1 by means of the *Salmonella typhimurium* assay (TA98 and TA100 strains), and some of them showed an efficient antimutagenic capacity: kaempferol, morin, fisetin, biochanin A, and rutin (Francis et al., 1989). Also, it was reported that kolaviron, a flavonoid from the seeds of *Garcinia kola* was able to inhibit the amount of micronuclei and the hepatic oxidative damage induced by

The determination of ammonia as antimutagen is included in this review considering that the chemical has been used as one of the agents to decontaminate AFB1; therefore, confirmation of its utility through genetic endpoints seems interesting. In the described report, mice were fed for four weeks with AFB1 contaminated corn and concomitantly treated with ammonium hydroxide (Marquez-Marquez et al., 1993). The results showed a significant reduction in the rate of micronucleated normochromatic erythrocytes starting from the first week of the assay, and at the fourth week of treatment the inhibition reached 60 %; besides, at the last week of the test, the quantification of SCE showed an inhibition of

Coffee is a beverage of habitual consumption that has shown controversial results concerning its genotoxic/antigenotoxic potential; however, there is an interesting study by Abraham (1991) who evaluated the inhibitory effect of standard instant coffee on the number of mice bone marrow micronuclei. Mice were orally administered coffee 2 and 20 h before injecting the carcinogen, and observations made at 28 and 48 h showed a dosedependent decrease in the rate of micronuclei, with a reduction of more than 60 % with the

In regard to constituents of apiaceous vegetables, such as carrots, parsnips, celery or parsley, Peterson et al. (2006) used a methoxyresorufin O-demethylase assay and a trp-recombination assay in *Saccharomyces cereviciae*, and found that 5-methoxypsoralen, and 8-methoxypsoralen reduced the CYP1A2-mediated mutagenesis of AFB1. In the same context, it was reported the hepatoprotective effect of ethanolic extract of *Phyllantus amarus* on AFB1-induced damage in mice, as well as the protective effect of soybean saponins against the aflatoxin in the *Salmonella typhimurium* assay, and a significant decrease of DNA-

adduct formation in human liver hepatoma cells (Jun et al., 2002; Naaz et al., 2007).

55 % in comparison with the level determined in the AFB1 treated group.

protective effect (Loarca-Piña et al., 1998; Cardador-Martinez et al., 2002).

and metabolic activation of AFB1.

AFB1 in rats (Farombi et al., 2005).

high tested dose (500 mg/kg).

In light of the serious effects that AFB1 contamination can originate, the authors agree that different socio-economical and toxicological approaches should be carried out for its elimination or control, including specific strategies regarding regulatory, supervisory, educational, scientific and technologic issues. Basic knowledge on the metabolism and the molecular and cellular fate of AFB1 is presently known, and various models have been used to test the effects of a number of chemopreventive agents, some of which have shown promising results, suggesting then, the pertinence of continuing with such strategy. However, it is reasonable to have a deeper knowledge on the chemical characteristics of each AFB1 metabolite, as well as on their interactions with macromolecules and cells, and to identify the more sensitive biomarkers for the assayed damage; this will be of help in designing more appropriate experimental projects, or clinical trials with the best candidates detected, in addition to preventing the selected genotoxic damage with more efficacy. At present, only a few agents have been tested in humans for evaluating their capacity of protection against AFB1 damage, although numerous chemicals have been evaluated in an almost isolated experimental form and have presented favorable results; therefore, extensive studies on these agents should be carried out so as to gain knowledge on their safety, efficacy, and mechanism of action, in order to select those more suitable for chemopreventive purposes.

#### **12. References**


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**15** 

*Qeshm Island* 

 *I. R. Iran* 

**Phytoinhibition of Growth and** 

*1Department of Biochemistry, Faculty of Medical Sciences,* 

*3Faculty of Basic Sciences, Shahed University, Tehran,* 

*Tarbiat Modares University, Tehran,* 

**Aflatoxin Biosynthesis in Toxigenic Fungi** 

*2Faculty of Medicine, Islamic Azad University, Qeshm International Branch,* 

Aflatoxins are primarily produced by the fungi *Aspergillus flavus* and *Aspergillus parasiticus*, which contaminate a wide variety of food and feed commodities including maize, oilseeds,

Presence of aflatoxins in food chain is associated with decrease in quality and quantity of food and feed materials. In addition, consumption of aflatoxin-contaminated products can pose a risk of development of various diseases in human and animals. Aflatoxins are produced in toxigenic fungi after undergoing biosynthesis pathway involving several enzymes and reactions. Upon consumption of aflatoxin contaminated products by human and animals, the toxin undergoes metabolism via cytochrome P450 enzymes in the liver. Aflatoxin metabolism in mammalian organs is a committed process and different metabolites are produced which can exert adverse effects of toxic metabolites. Aflatoxin epoxide (8,9-epoxide) is the major toxic metabolite which can bind to DNA and induce hepatocellular carcinoms. The extent of aflatoxin toxicity and carcinogenicity in human and animals depends on several factors including the metabolic capacity of the organism. Aflatoxin contamination of food products is associated with health and socioeconomic costs which is difficult to valuate in the developing countries. Moreover, the current regulations do little to help reduce aflatoxin and related health effects. Therefore the focus should be on promoting the adaptation of strategies that can control aflatoxin and its associated health risks. According to Wu and Khlangwiset (2010), interventions to reduce aflatoxin-induced illness can be grouped into three categories; agricultural, dietary and clinical. Agricultural interventions are methods that can be applied either in the field (preharvest) or in drying, storage and transportation (postharvest) to reduce aflatoxin levels in food. The dietary and clinical interventioans are considered as secondary interventions by which the aflatoxinrelated illness can be reduced. These two types interventions are associated with advantages

Due to concern for the potential effects of aflatoxins on human health, most countries have legislation that restricts marketing of aflatoxin-contaminated grains [Van Egmond, 1989]. The United States Food and Drug Administration has set an aflatoxin limit of 20 µg/kg for

spices, groundnuts, tree nuts, milk and dried fruits [Strosnider et al., 2006].

**1. Introduction** 

and disadvantages.

Abdolamir Allameh1, Tahereh Ziglari2 and Iraj Rasooli3

